Eye-Drop Vaccine Containing Copolymer 1 for Therapeutic Immunization

ABSTRACT

The invention provides an eye-drop vaccine for therapeutic immunization of a mammal comprising Copolymer 1, a Copolymer 1-related peptide, or a Copolymer 1-related polypeptide, for treating neuronal degeneration caused by an injury or disease, disorder or condition in the central nervous system (CNS) or peripheral nervous system (PNS), for preventing or inhibiting neuronal secondary degeneration which may otherwise follow primary injury in the CNS, for promoting nerve regeneration in the CNS or in the PNS after an injury, disease, disorder or condition or for protecting CNS and PNS cells from glutamate toxicity.

FIELD OF THE INVENTION

The present invention is in the field of Immunology and relates to aneye-drop vaccine comprising a random copolymer, in particular Copolymer1, a Copolymer 1-related peptide, or a Copolymer 1-related polypeptide,as the active agent, and to a method of therapeutic immunization of amammal, in particular for neuroprotection in the central nervous system(CNS) or in the peripheral nervous system (PNS) after an injury,disease, disorder or condition or for protecting CNS and PNS cells fromglutamate toxicity.

Abbreviations: BSA: bovine serum albumin; CFA: complete Freund'sadjuvant; CNS: central nervous system; Cop 1: Copolymer 1, glatirameracetate; FCS: fetal calf serum; IFA: incomplete Freund's adjuvant; IOP:intraocular pressure; MBP: myelin basic protein; NS: nervous system;PBS: phosphate-buffered saline; PNS: peripheral nervous system; RGC:retinal ganglion cells.

BACKGROUND OF THE INVENTION

The nervous system comprises the central and the peripheral nervoussystem. The peripheral nervous system (CNS) is composed of the brain andspinal cord; the peripheral nervous system (PNS) consists of all of theother neural elements, namely the nerves and ganglia outside of thebrain and spinal cord.

Damage to the nervous system may result from a traumatic injury, such aspenetrating trauma or blunt trauma, or a disease, disorder or condition,including but not limited to Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, diabeticneuropathy, glaucoma, senile dementia, and ischemia.

Neurodegenerative disorders are commonly associated with ongoingneuronal loss in the CNS. Following the loss of neurons caused byprimary risk factors, additional (“secondary”) neuronal loss is mediatedby self-compounds, such as glutamate, nitric oxide or reactive oxygenspecies, that exceed their physiological concentrations. These compoundsare implicated in various types of neurological disorders and acute CNSinjuries. It is interesting to note that the destructive componentscommon to neurodegenerative diseases have also been identified inautoimmune diseases such as multiple sclerosis; in this disease, myelindamage in the CNS is accompanied by subsequent neuronal loss.

Glaucoma is a slow-progressing optic neuropathy with a high incidence inthe elderly population (approximately 1%). Until recently, it wasassociated with high intraocular pressure (IOP) and therefore attemptshave been focused on slowing down the disease progression byanti-hypertensive drugs. Over the years, it became apparent thatglaucoma is a family of diseases and not all are associated withpressure. Moreover, it became clear that even when the disease isassociated with pressure, the latter may be reduced to normal and evenbelow normal values and degeneration may continue. An ongoing discussionamong clinicians has questioned whether the continuous degeneration inglaucomatous patients, in spite of normal IOP values, is a reflection ofthe existence of additional risk factors besides pressure or areflection of the increased vulnerability of the remaining neurons andfibers and thus the need to reduce IOP below normal values.

We have suggested in 1996 that the mechanism underlying progressive lossof vision in glaucoma is similar to that occurring in any acute insultto the nervous system or any neurodegenerative disease of the CNS(Schwartz et al, 1996). According to this proposal, in addition to theprimary risk factor, e.g. pressure, there is an ongoing process ofdegeneration that affects neurons that spared the primary event(Schwartz et al, 1996; Schwartz and Yoles, 2000a and 2000b). Thisprocess is mediated by compounds that emerged as a result of the primaryevent or by deficit as a result of the primary risk factor, all of whichcreate a hostile environment to neurons adjacent to the primary insult.

We have further recently observed that under neurodegenerativeconditions caused by mechanical (axotomy) or biochemical (glutamate,oxidative stress) insults, the immune system plays a critical role.Thus, it has been found that activated T cells that recognize an antigenof the nervous system (NS) promote nerve regeneration or conferneuroprotection, as described for example in PCT Publication No. WO99/60021. More specifically, T cells reactive to myelin basic protein(MBP) were shown to be neuroprotective in rat models of partiallycrushed optic nerve (Moalem et al., 1999) and of spinal cord injury(Hauben et al., 2000). Until recently, it had been thought that theimmune system excluded immune cells from participating in nervous systemrepair. It was quite surprising to discover that NS-specific activated Tcells could be used to promote nerve regeneration or to protect nervoussystem tissue from secondary degeneration which may follow damage causedby injury or disease of the CNS or PNS.

It was further observed by the present inventors that stressfulconditions in the CNS harness the adaptive immune response to cope withthe stress and that this response is genetically controlled. Thus, thesurvival rate of retinal ganglion cells (RGCs) in adult mice or ratsafter crush injury of the optic nerve or intravitreal injection of atoxic dosage of glutamate was shown to be up to two-fold higher instrains that are resistant to CNS autoimmune diseases than insusceptible strains. The difference was found to be attributable to abeneficial autoimmune T cell response that was spontaneously evokedafter CNS insult in the resistant, but not in susceptible, strains.Thus, the survival rate of neurons as a result of such an insult ishigher when T cell response directed against self is evoked, providedthat it is well-regulated. In other words, it was demonstrated that aprotective autoimmune response is evoked to oppose the stressfulconditions so as to protect the animal from the insult consequences. Itwas further observed that in animals with an impaired ability toregulate such a response, or in animals devoid of mature T cells (as aresult of having undergone thymectomy at birth), the ability to copewith the stressful conditions is reduced. Consequently, the survivalrate of neurons following CNS insult in these animals is significantlylower than in animals endowed with an effective mechanism for mountingprotective autoimmune T cell-mediated response (Kipnis et al., 2001).

More recent studies in our laboratory have shown that autoimmuneneuroprotection is the body's physiological defense mechanism awakenedwhen CNS injury occurs (Kipnis et al., 2001; Yoles et al., 2001). Wedemonstrated that resistance to increased IOP differs among strains(Bakalash et al., 2002) and that this difference is linked to theability to harness an autoimmune response with a beneficial outcome. Wefurther showed that in the absence of mature T cells (through neonatalthymectomy), the relative resistance to IOP elevation loses itsbeneficial trait, and vice versa, when splenocytes from a resistantstrain are passively transferred to an MHC-matched susceptible strain,the neuroprotective effect is resumed (Bakalash et al., 2002). It wasfurther shown by our group that passive vaccination with T cells is alsoeffective in acute injuries such as partial optic nerve crush or spinalcord contusion (Kipnis et al., 2001; Moalem et al., 1999).

Attempting to boost such an anti-self response as a way of protectingneurons from insulting conditions has revealed that the vaccinatingantigen should be derived from compounds residing in the site of thelesion. Thus, the use of the self-antigen derived frominterphotoreceptor binding protein (IRBP), the most abundant peptide inthe eye (Bakalash et al., 2002; Mizrahi et al., 2002), resulted in RGCprotection in both susceptible and resistant strains. In contrast, theuse of peptides derived from compounds residing in the myelin associatedwith the optic nerve led to no benefit to the retinal ganglion cellssuffering from IOP elevated insult.

Trying to design a vaccination for glaucoma that will boost the immunesystem without risk of evoking an autoimmune disease, we chose to focuson Copolymer 1, and have shown that it is neuroprotective for glaucomawhen given with an adjuvant (Bakalash et al., 2002; Schori et al., 2001;Schwartz and Kipnis, 2002; WO 01/52878; WO 01/93893). Cop-1immunologically cross-reacts with a wide variety of self-reactive Tcells. Accordingly, its activity is reminiscent of that of alteredpeptide ligand, a self-peptide that has been altered and has lostpathogenicity as a result (WO 02/055010; Kipnis and Schwartz, 2002).

Copolymer 1, also called Cop 1 or glatiramer acetate, is anon-pathogenic synthetic random copolymer composed of the four aminoacids: L-Glu, L-Lys, L-Ala, and L-Tyr, with an average molecularfraction of 0.141, 0.338, 0.427, and 0.095, respectively, and an averagemolecular weight of 4,700-11,000. COPAXONE® (a trademark of TevaPharmaceutical Industries Ltd., Petach Tikva, Israel), the brand namefor glatiramer acetate, is currently an approved drug in many countriesfor the treatment of multiple sclerosis. It is very well tolerated withonly minor adversary reactions. Although treatment with Cop 1 byingestion or inhalation is disclosed in U.S. Pat. No. 6,214,791, thesole route of administration of Cop 1 to multiple sclerosis patients isby daily subcutaneous injection.

Recently it was found that in animal models Cop 1 provides a beneficialeffect for several additional disorders. Thus, Cop 1 suppresses theimmune rejection manifested in graft-versus-host disease (GVHD) in caseof bone marrow transplantation (Schlegel et al., 1996; U.S. Pat. No.5,858,964), as well as in graft rejection in case of solid organtransplantation (Aharoni et al., 2001). Cop 1 and Cop 1-relatedcopolymers and peptides have been disclosed in WO 00/05250 for treatingautoimmune diseases.

WO 01/52878 and WO 01/93893 of the present applicants disclose that Cop1, Cop 1-related peptides and polypeptides and T cells activatedtherewith protect CNS cells from glutamate toxicity and prevent orinhibit neuronal degeneration or promote nerve regeneration in the CNSand PNS. WO 01/93828 discloses that Cop 1 can be used for treatment ofCNS disorders. None of these publications discloses immunization byadministration of eye-drops containing Cop 1.

Poultry vaccines for administration as eye drops comprising a live virusor recombinant DNA coding for immunogenic proteins from infectiousagents have been described for prevention of viral diseases in aviananimals (Mukibi-Muka et al., 1984; Sharma, 1999; Russell and Mackie,2001).

Citation or identification of any reference in this section or any otherpart of this application shall not be construed as an admission thatsuch reference is available as prior art to the invention.

SUMMARY OF THE INVENTION

It has now been found, in accordance with the present invention, thatCopolymer 1, when administered as an eye-drop vaccine, evokes a systemicT cell-dependent immune response needed for neuroprotection in the CNSor PNS, as exemplified by protection of retinal ganglion cells (RGCs)against death induced by acute or chronic intraocular pressure (IOP)elevation.

The present invention thus relates, in one aspect, to an eye-dropvaccine comprising an active agent selected from the group consisting ofCopolymer 1, a Copolymer 1 related-peptide, and a Copolymer 1-relatedpolypeptide.

In another aspect, the present invention relates to the use of an activeagent selected from the group consisting of Copolymer 1, a Copolymer 1related-peptide, and a Copolymer 1-related polypeptide, for themanufacture of an eye-drop vaccine.

The eye-drop vaccine according to the invention is particularly usefulfor therapeutic immunization of a mammal, in particular humans, forneuroprotection for treating neuronal degeneration caused by an injury,disease, disorder or condition in the central nervous system (CNS) orperipheral nervous system (PNS), for preventing or inhibiting neuronalsecondary degeneration which may otherwise follow a primary injury inthe CNS, for promoting nerve regeneration in the CNS or in the PNS afteran injury, disease, disorder or condition, or for protecting CNS and PNScells from glutamate toxicity.

In a further aspect, the present invention provides a method oftherapeutic immunization for treating neuronal degeneration caused by aninjury, disease, disorder or condition in the central nervous system(CNS) or peripheral nervous system (PNS), for preventing or inhibitingneuronal secondary degeneration which may otherwise follow a primaryinjury in the CNS, for promoting nerve regeneration in the CNS or in thePNS after an injury, disease, disorder or condition or for protectingCNS and PNS cells from glutamate toxicity, which comprises immunizing anindividual in need with an eye-drop vaccine comprising an active agentselected from the group consisting of Copolymer 1, a Copolymer 1-relatedpeptide, and a Copolymer 1-related polypeptide, in an amount effectiveto treat, prevent or inhibit said neuronal degeneration caused by saidinjury, disease, disorder or condition in the individual.

In the eye-drop vaccine of the invention, the active agent may beadministered without any adjuvant, for example in saline orphosphate-buffered saline (PBS), or it may be administered with asoluble adjuvant such as a cytokine, e.g. IL-2, IL-12, GM-CSF or IFN-γ,and the like.

In the most preferred embodiment, the active agent of the eye-dropvaccine of the invention is Copolymer 1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that Copolymer 1 (Cop-1) has a long-lasting effect inprotecting RGCs from IOP-induced death in a chronic model.

FIGS. 2A-2B show that immunization with 500 μg Cop-1 without adjuvant(2A) at the day 7 after the first laser (2B) protects RGCs fromIOP-induced RGC death in a chronic model.

FIGS. 3A-3B show that the effect of Cop-1 is T cell-dependent. Treatmentof elevated IOP in non-thymectomized animals with Cop-1 was moreeffective than in thymectomized animals (3A) and more effective than theglaucoma drug brimonidine (3B).

FIGS. 4A-4B show that Cop-1 applied in eye drops protects fromIOP-induced RGC death in a chronic model. Five drops of 1 mg Cop-1 eachgiven at 5-minute intervals were applied immediately (4A) or 7 days (4B)after IOP elevation in a chronic model of IOP elevation. Retinas wereexcised 3 weeks later.

FIGS. 5A-5B show that Cop-1 protects RGCs against acute transient IOPelevation when administered subcutaneously (5A) or as eye drops (5B).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “Copolymer 1”, “Cop 1”, “Cop-1”, and“glatiramer acetate” are each used interchangeably.

For the purpose of the present invention, “Cop 1 or a Cop 1-relatedpeptide or polypeptide” is intended to include any peptide orpolypeptide, including a random copolymer, that cross-reactsfunctionally with myelin basic protein (MBP) and is able to compete withMBP on the MHC class II in the antigen presentation.

A copolymer for use as active agent in the eye-drop vaccine of thepresent invention may be a random copolymer comprising a suitablequantity of a positively charged amino acid such as lysine (K) orarginine (R), in combination with a negatively charged amino acid(preferably in a lesser quantity) such as glutamic acid (E) or asparticacid (D), optionally in combination with a non-charged neutral aminoacid such as alanine (A) or glycine (G), serving as a filler, andoptionally with an amino acid adapted to confer on the copolymerimmunogenic properties, such as an aromatic amino acid like tyrosine (Y)or tryptophan (W).

The copolymers for use in the present invention can be composed of L- orD-amino acids or mixtures thereof. As is known by those of skill in theart, L-amino acids occur in most natural proteins. However, D-aminoacids are commercially available and can be substituted for some or allof the amino acids used to make the copolymers used in the presentinvention. The present invention contemplates the use of copolymerscontaining both D- and L-amino acids, as well as copolymers consistingessentially of either L- or D-amino acids.

In one embodiment, the active agent for use in the present inventioncomprises at least one random three- or four-amino acid copolymercomprising one amino acid selected from each of the four followinggroups: (a) lysine (K) and arginine (R); (b) glutamic acid (E) andaspartic acid (D); (c) alanine (A) and glycine (G); and (d) tyrosine (Y)and tryptophan (W).

In one preferred embodiment, the copolymer comprises a combination ofthe amino acids tyrosine, glutamic acid, alanine, and lysine, hereindesignated poly-YEAK, of net overall positive electrical charge, and ismost preferably Copolymer 1, of the following molar ratio of the aminoacids: about 0.14 glutamic acid, about 0.43 alanine, about 0.10tyrosine, and about 0.34 lysine. It may be a low molecular weight orhigh molecular weight copolymer being a polypeptide from about 15 toabout 100, preferably from about 40 to about 80, amino acids in length.The copolymer has an average molecular weight of about 2,000-40,000 Da,preferably of about 2,000-13,000 Da, more preferably of about4,700-13,000 Da, most preferably of about 5,000-9,000 Da, and mostlypreferred of about 6,000-8,000 Da. This preferred copolymer, Cop 1, ismost preferably in the form of its acetate salt known under the genericname glatiramer acetate. Preferred molecular weight ranges and processesfor making a preferred form of Cop 1 are described in U.S. Pat. No.5,800,808, the entire contents of which are hereby incorporated byreference in their entirety as if fully disclosed herein.

It is clear that this is given by way of example only, and that theactive agent can be varied both with respect to the constituents andrelative proportions of the constituents, thus obtaining poly-YEAKcopolymers different from Cop 1.

In another embodiment, the active agent of the eye-drop vaccine of theinvention is a Cop 1-related polypeptide that is a random copolymercontaining four different amino acids, each from a different one of thegroups (a) to (d), but excluding Cop 1. The activity exhibited byCopolymer 1 is expected to remain if one or more of the followingsubstitutions is made in the amino acid composition of the copolymer:aspartic (D) acid for glutamic acid (E), glycine (G) for alanine (A),arginine (R) for lysine (K), and tryptophan (W) for tyrosine (Y).

Thus, in another embodiment, the Cop 1-related polypeptide of theinvention may include any of those copolymers disclosed in WO 00/05250,the entire contents of which being hereby incorporated herein byreference as if fully disclosed herein, and other synthetic amino acidcopolymers such as the random four-amino acid copolymers described byFridkis-Hareli et al. (2002) as candidates for treatment of multiplesclerosis, namely copolymers (14-, 35- and 50-mers) containing the aminoacids phenylalanine, glutamic acid, alanine and lysine (poly-FEAK), ortyrosine, phenylalanine, alanine and lysine (poly-YFAK), and any othersimilar copolymer to be discovered that can be considered a universalantigen similar to Cop 1.

In another embodiment, the Cop 1-related polypeptide of the invention isa copolymer containing a combination of three different amino acids eachfrom a different one of three groups of the groups (a) to (d). Thesecopolymers are herein referred to as terpolymers. In a more preferredembodiment, the mole fraction of amino acids of the terpolymers is aboutwhat is preferred for Copolymer 1.

In one embodiment, the terpolymers for use in the present inventioncontain tyrosine (Y), alanine (A), and lysine (K), hereinafterdesignated poly-YAK. The average molar fraction of the amino acids inthese terpolymers can vary. For example, tyrosine can be present in amole fraction of about 0.005-0.250; alanine can be present in a molefraction of about 0.3-0.6; and lysine can be present in a mole fractionof about 0.1-0.5, but preferably the molar ratios of tyrosine, alanineand lysine are about 0.10 to about 0.54 to about 0.35. The averagemolecular weight of poly-YAK is about 2,000-40,000 Da, preferably about3,000-35,000 Da, more preferably about 5,000-25,000 Da. It is possibleto substitute arginine (R) for lysine (K), glycine (G) for alanine (A),and/or tryptophan (W) for tyrosine (Y).

In another embodiment, the terpolymers for use in the present inventioncontain tyrosine (Y), glutamic acid (E), and lysine (K), hereinafterdesignated poly-YEK. The average mole fraction of the amino acids inthese terpolymers can vary: glutamic acid can be present in a molefraction of about 0.005-0.300, tyrosine can be present in a molefraction of about 0.005-0.250, and lysine can be present in a molefraction of about 0.3-0.7, but preferably the molar ratios of glutamicacid, tyrosine, and lysine are about 0.26 to about 0.16 to about 0.58.The average molecular weight of poly-YEK is about 2,000-40,000 Da,preferably about 3,000-35,000 Da, more preferably about 5,000-25,000 Da.It is possible to substitute arginine (R) for lysine (K), aspartic acid(D) for glutamic acid (E), and/or tryptophan (W) for tyrosine (Y).

In a further embodiment, the terpolymers for use in the presentinvention contain lysine (K), glutamic acid (E), and alanine (A),hereinafter designated poly-KEA. The average molar fraction of the aminoacids in these polypeptides can also vary. For example, glutamic acidcan be present in a mole fraction of about 0.005-0.300, alanine in amole fraction of about 0.005-0.600, and lysine can be present in a molefraction of about 0.2-0.7, but preferably the molar ratios of glutamicacid, alanine and lysine are about 0.15 to about 0.48 to about 0.36. Theaverage molecular weight of YEK is about 2,000-40,000 Da, preferablyabout 3,000-35,000 Da, more preferably about 5,000-25,000 Da. It ispossible to substitute arginine (R) for lysine (K), aspartic acid (D)for glutamic acid (E), and/or glycine (G) for alanine (A).

In still another embodiment, the terpolymers for use in the presentinvention contain tyrosine (Y), glutamic acid (E), and alanine (A),hereinafter designated poly-YEA. The average molar fraction of the aminoacids in these polypeptides can vary. For example, tyrosine can bepresent in a mole fraction of about 0.005-0.250, glutamic acid can bepresent in a mole fraction of about 0.005-0.300, and alanine can bepresent in a mole fraction of about 0.005-0.800, but preferably themolar ratios of glutamic acid, alanine, and tyrosine are about 0.21 toabout 0.65 to about 0.14. The average molecular weight of poly-YEA isabout 2,000-40,000 Da, preferably about 3,000-35,000 Da, and morepreferably about 5,000-25,000 Da. It is possible to substitutetryptophan (W) for tyrosine (Y), aspartic acid (D) for glutamic acid(E), and/or glycine (G) for alanine (A).

The terpolymers can be made by any procedure available to one of skillin the art, for example as described in the above-mentioned publicationsWO 01/52878 and WO 01/93893.

As binding motifs of Cop 1 to MS-associated HLA-DR molecules are known,polypeptides of fixed sequence can readily be prepared and tested forbinding to the peptide-binding groove of the HLA-DR molecules asdescribed in Fridkis-Hareli et al. (1999). Examples of such peptides arethose disclosed in WO 005249, the entire contents of which are herebyincorporated by reference as if fully disclosed herein. Thirty-two ofthe peptides specifically disclosed in said application are reproducedin Table I hereinbelow (SEQ ID NO:1 to NO:32). These are 15-mer peptidescomprising the 4 amino acids alanine, glutamic acid, lysine and tyrosine(peptides 2, 3, 5-32) or only the 3 amino acids alanine, lysine andtyrosine (peptides 1, 4). Such peptides and other similar peptides wouldbe expected to have similar activity as Cop 1 and are encompassed withinthe definition of Cop 1-related of the invention. TABLE 1 SEQ ID NO.Peptide Sequence  1 AAAYAAAAAAKAAAA  2 AEKYAAAAAAKAAAA  3AKEYAAAAAAKAAAA  4 AKKYAAAAAAKAAAA  5 AEAYAAAAAAKAAAA  6 KEAYAAAAAAKAAAA 7 AEEYAAAAAAKAAAA  8 AAEYAAAAAAKAAAA  9 EKAYAAAAAAKAAAA 10AAKYEAAAAAKAAAA 11 AAKYAEAAAAKAAAA 12 EAAYAAAAAAKAAAA 13 EKKYAAAAAAKAAAA14 EAKYAAAAAAKAAAA 15 AEKYAAAAAAAAAAA 16 AKEYAAAAAAAAAAA 17AKKYEAAAAAAAAAA 18 AKKYAEAAAAAAAAA 19 AEAYKAAAAAAAAAA 20 KEAYAAAAAAAAAAA21 AEEYKAAAAAAAAAA 22 AAEYKAAAAAAAAAA 23 EKAYAAAAAAAAAAA 24AAKYEAAAAAAAAAA 25 AAKYAEAAAAAAAAA 26 EKKYAAAAAAAAAAA 27 EAKYAAAAAAAAAAA28 AEYAKAAAAAAAAAA 29 AEKAYAAAAAAAAAA 30 EKYAAAAAAAAAAAA 31AYKAEAAAAAAAAAA 32 AKYAEAAAAAAAAAA

The present invention relates, in one aspect, to an eye-drop vaccinecomprising an active agent selected from the group consisting ofCopolymer 1, a Copolymer 1 related-peptide, and a Copolymer 1-relatedpolypeptide.

In another aspect, the present invention relates to the use of an activeagent selected from the group consisting of Copolymer 1, a Copolymer 1related-peptide, and a Copolymer 1-related polypeptide for themanufacture of an eye-drop vaccine.

In one embodiment, the eye-drop vaccine comprises the active agentdissolved in any suitable carrier such as saline or PBS, without anyadjuvant.

In another embodiment, the eye-drop vaccine comprises the active agenttogether with a suitable soluble adjuvant such as a soluble cytokine asexemplified, but not limited to, the cytokines IL-2, IL-12, GM-CSF orIFN-γ.

The present invention further relates to a method of therapeuticimmunization for neuroprotection which comprises immunizing anindividual in need with an eye-drop vaccine comprising an active agentselected from the group consisting of Copolymer 1, a Copolymer 1related-peptide, and a Copolymer 1-related polypeptide, in an amounteffective to afford neuroprotection to said individual.

In one embodiment, the invention provides a method of therapeuticimmunization for treating neuronal degeneration caused by an injury inthe CNS or PNS, which comprises immunizing an individual in need with aneye-drop vaccine comprising an active agent selected from the groupconsisting of Copolymer 1, a Copolymer 1 related-peptide, and aCopolymer 1-related polypeptide, in an amount effective to treat theneuronal degeneration caused by the injury in said individual.

In another embodiment, the invention provides a method of therapeuticimmunization for preventing or inhibiting neuronal secondarydegeneration which may otherwise follow a primary injury in the CNS,which comprises immunizing an individual in need with an eye-dropvaccine comprising an active agent selected from the group consisting ofCopolymer 1, a Copolymer 1 related-peptide, and a Copolymer 1-relatedpolypeptide, in an amount effective for preventing or inhibiting theneuronal degeneration which may follow a primary injury in the CNS ofsaid individual.

In a further embodiment, the invention provides a method of therapeuticimmunization for promoting nerve regeneration in the CNS or in the PNSafter an injury, which comprises immunizing an individual in need withan eye-drop vaccine comprising an active agent selected from the groupconsisting of Copolymer 1, a Copolymer 1 related-peptide, and aCopolymer 1-related polypeptide, in an amount effective for promotingnerve regeneration in the CNS or in the PNS of said individual after theinjury.

Any injury in the CNS or PNS can be treated according to the inventionsuch as, but not limited to, spinal cord injury, blunt trauma,penetrating trauma, brain coup or contrecoup, hemorrhagic stroke, andischemic stroke.

In yet another embodiment, the invention relates to a method oftherapeutic immunization for treating neuronal degeneration caused by adisease, disorder or condition in the CNS or PNS, which comprisesimmunizing an individual in need with an eye-drop vaccine comprising anactive agent selected from the group consisting of Copolymer 1, aCopolymer 1 related-peptide, and a Copolymer 1-related polypeptide, inan amount effective to treat the neuronal degeneration caused by thedisease, disorder or condition in the CNS or PNS of said individual.

In yet a further embodiment, the invention relates to a method oftherapeutic immunization for promoting nerve regeneration in the CNS orin the PNS after a disease, disorder or condition, which comprisesimmunizing an individual in need with an eye-drop vaccine comprising anactive agent selected from the group consisting of Copolymer 1, aCopolymer 1 related-peptide, and a Copolymer 1-related polypeptide, inan amount effective to promote nerve regeneration in the CNS or in thePNS needed following a disease, disorder or condition of the CNS or PNSin said individual.

In still a further embodiment, the invention relates to a method oftherapeutic immunization for protecting CNS and PNS cells from glutamatetoxicity, which comprises immunizing an individual in need with aneye-drop vaccine comprising an active agent selected from the groupconsisting of Copolymer 1, a Copolymer 1 related-peptide, and aCopolymer 1-related polypeptide, in an amount effective to protect CNSor PNS cells in said individual from glutamate toxicity.

Among the diseases, disorders and conditions that may be treatedaccording to the invention are, without being limited to, a seniledementia including Alzheimer's disease, a Parkinsonian syndromeincluding Parkinson's disease, facial nerve (Bell's) palsy, Huntington'schorea, a motor neuron disease including amyotrophic lateral sclerosis,a prion disease including Creutzfeldt-Jakob disease, Alper's disease,Batten disease, Cockayne syndrome, Lewy body disease, statusepilepticus, carpal tunnel syndrome, intervertebral disc herniation,vitamin deficiency such as vitamin B deficiency, epilepsy, amnesia,anxiety, hyperalgesia, psychosis, seizures, oxidative stress, opiatetolerance and dependence, an autoimmune disease such as multiplesclerosis (MS), or a peripheral neuropathy associated with a diseasesuch as amyloid polyneuropathy, diabetic neuropathy, uremic neuropathy,porphyric polyneuropathy, hypoglycemia, Sjogren-Larsson syndrome, acutesensory neuropathy, chronic ataxic neuropathy, biliary cirrhosis,primary amyloidosis, obstructive lung diseases, acromegaly,malabsorption syndromes, polycythemia vera, IgA and IgG gammapathies,complications of various drugs such as nitrofurantoin, metronidazole,isoniazid and toxins such as alcohol or organophosphates,Charcot-Marie-Tooth disease, ataxia telangiectasia, Friedreich's ataxia,adrenomyeloneuropathy, giant axonal neuropathy, Refsum's disease,Fabry's disease, lipoproteinemia, non-arteritic optic neuropathy,age-related macular degeneration, a retinal disorder such as retinaldegeneration or a disease associated with abnormally elevatedintraocular pressure such as glaucoma.

As mentioned in the Background of the Invention section, we havesuggested in 1996 that perhaps in glaucoma, like in any acute insult tothe nervous system or any neurodegenerative disease of the CNS, there isan ongoing process of degeneration that affects neurons that spared theprimary event, e.g. pressure elevation (Schwartz et al, 1996). Thisprocess is mediated by compounds that emerged as a result of the primaryevent or by deficit as a result of the primary risk factor, all of whichcreate a hostile environment to neurons adjacent to the primary insult(Schwartz et al, 1996; Schwartz and Yoles, 2000a and 2000b). Recognitionthat such processes may take place in glaucoma, encouraged us to searchfor a therapy that, if given at any time, it may stop the progression ofdegeneration.

Indeed, since 1996 attempts are being made worldwide to identifymediators of toxicity to find ways that block, circumvent or eliminatethese mediators, or to find ways to make the remaining neurons moretolerable to the hostile milieu crated by the ones that have alreadydegenerated.

Our group discovered about four years ago that injured optic nerve ordamaged retina deliver stress signal to the systemic immune system andrecruit its help by leading to an evocation of T cell response directedagainst abundant antigens residing in the site of the stress. The immuneresponse specificity is a way to bring T cells to the site of thelesion. Once activated, such T cells serve as a source of cytokines andneurotrophic factors affecting locally microglia, astrocytes and perhapseven neurons. In the course of our studies we realized that such animmune response is basically needed to help the body to cope with theinjurious conditions and in disease conditions, like in trauma, need tobe boosted (Moalem et al., 1999; Schori et al., 2001 and 2002; Kipnis etal., 2000).

We have discovered several ways that successfully boost the Tcell-dependent immune response needed to protect damaged nerve from itshostile environment. A risk-free protection was found by the use of acompound that cross-reacts with a low affinity with a wide spectrum ofself-antigens. One such compound is Copolymer 1 or glatiramer acetate.This compound has an enormous advantage that it is an approved drug formultiple sclerosis patients and thus does not carry the risk of causingan autoimmune disease (Schori et al., 2001 and 2002; Kipnis et al.,2000).

As described in our previous applications WO 01/52878 and WO 01/93893,Copolymer 1 emulsified in adjuvant was found to be protective for RGCsagainst elevated IOP in a chronic model of elevated IOP. Here wediscovered that Cop-1 is strong enough and thus can evoke an intensiveimmune response that can protect effectively against consequences of IOPin both acute and chronic models of IOP. Interestingly, Cop-1, whenadministered in a regimen used for chronic MS disease, i.e. dailyrepeated injection, wipes out the benefit of a single injection,substantiating the contention that the requirements for autoimmunedisease and for neurodegenerative disease are different (Schwartz andKipnis. 2002; Kipnis and Schwartz, 2002). While the former needssuppression and the latter needs immune activation, the two may be metif one takes an approach of immunomodulation.

Another feature which became clear in the experiments of the presentapplication is that intervention, in cases of RGC loss associated withchronic high IOP, is effective at any time in stopping diseaseprogression. The fact that in the chronic model of IOP, interaction wasmore effective on day 7 than on day 0, do suggest that RGC loss is notinitiated immediately after the pressure elevation, unlike in the acutemodel, in which the elevation of IOP is so high that death occurs muchearlier.

That protection by vaccination with Cop-1 is not mediated by the drugCop-1 itself, but by the immune response that it evokes, became evidentwhen no effect was observed in T cell deprived animals. This explainswhy there is no need for daily administration of the drug forneuroprotection, as is often the case with pharmacological compoundsthat its clearance from the body is fast and there is a need forsustaining the drug. Administration of the compound, however, is neededperiodically to sustain optimal level of T cells needed for theprotection from the ongoing degeneration.

The way by which Cop-1 is protective in glaucoma is by evoking T cellresponse. The evoked T cells home to the eye in which they encountermicroglia that can present the same self-antigen residing in the eyewith which they can cross-react. The activated T cells are the source ofcytokine and neurotrophic factors needed for the protection. Our invitro studies have suggested that activated T cells can upregulatecluster of genes in microglia that can cope with stress and also genesthat are associated with their buffering activity.

Other studies revealed that compound that can neutralize toxic compoundin the eye could potentially be developed as a therapy for glaucoma.Among such compounds are NMDA antagonists, NO synthetase inhibitors. Asglaucoma like any other neurodegenerative disease is not a singlecompound disease it is suggestive that in order to get a globalprotection one has to combine several drugs. The advantage of thevaccination according to the invention is in the fact that it simulatesthe body's own way of getting rid of stress and it invokes activity ofthe immune cells that is site-specific, but not insult-specific, andthus can protect from a wide range of threats.

According to the present invention, a single treatment with Cop 1 eyedrops was found as an effective therapy in protecting RGCs againstIOP-induced RGC loss in acute and chronic glaucoma rat models. In viewof the previous publications by the inventors showing theneuroprotective effect of Cop 1 when injected to the animals, it wasvery surprising to discover that Cop 1 confers neuroprotectionsystemically when administered as eye drops.

In one preferred embodiment of the invention, the eye-drop vaccinecomprising Copolymer 1, a Copolymer 1-related peptide or a Copolymer1-related polypeptide is used to treat glaucoma, namely to arrestprogression of glaucoma, a neurodegenerative disease of the optic nerve.As mentioned before, elevation of intraocular pressure (IOP) is oftenassociated with chronic or acute glaucoma. However, very often,reduction of pressure is insufficient to stop disease progression.Elements of self-destruction are found to be associated with the diseaseprogression. Here we show that a single vaccination with Cop 1, given ineye drops (or subcutaneously, for comparison) is sufficient to rescueRGCs from IOP-induced loss. Moreover, we found that in the chronic modelof the disease, delayed treatment (7 days) is as effective as immediatetreatment in the chronic model of IOP. Evidence is provided thattreatment, either with the eye drops or systemically, is not directlyeffective but is immune-mediated. No effect is achieved in animalsdeprived of T cells, however the eye-drop effect is achieved when givencontralaterally to the side with the elevated pressure in the eye. Theresults and route of immunization can be implemented immediately forclinical development.

According to the present invention, the preferred copolymer for use asthe active agent of the invention is Cop 1, most preferably in the formof its acetate salt known under the generic name glatiramer acetate. Thedosage of Cop 1 to be administered will be determined by the physicianaccording to the age of the patient and stage of the disease and may bechosen from a range of 0.1 to 1,000 mg, preferably from a range of 10-80mg, more preferably 20-60 mg, although any other suitable dosage isencompassed by the invention.

For multiple sclerosis patients, the administration may be made daily inone or more doses, preferably from one to three daily doses in a totalof 0.1 to 1,000 mg, preferably within a range of 10-80 mg, morepreferably 20-60 mg, or in alternate days, but any other suitable dosageis envisaged by the invention according to the condition of the patient.For non-multiple sclerosis patients, the dosage of Cop 1 is as indicatedabove in a periodical frequency, e.g. at least once a week, to at leastonce a month or at least once every 2 or 3 months, or less frequently,but any other suitable interval between the immunizations is envisagedby the invention according to the condition of the patient.

The following examples illustrate certain features of the presentinvention but are not intended to limit the scope of the presentinvention.

EXAMPLES

Materials and Methods

(i) Animals. Inbred adult male Lewis and Sprague-Dawley (SPD) rats (8weeks; average weight 300 g) were supplied by the Animal Breeding Centerat The Weizmann Institute of Science (Rehovot, Israel). The rats weremaintained in a light- and temperature-controlled room and were matchedfor age and weight before each experiment. All animals were handledaccording to the regulations formulated by IACUC (International AnimalCare and Use Committee).

(ii) Induction of chronically high intraocular pressure (IOP). Blockageof aqueous outflow causes an increase in IOP, which results in RGC death(Bakalash et al., 2002; Schori et al., 2001; Jia et al., 2000a;Levkovitch-Verbin et al., 2002). An increase in IOP was achieved in theright eyes of rats as follows. Rats were deeply anesthetized byintramuscular injection of ketamine hydrochloride (50 mg/kg) andxylazine hydrochloride (0.5 mg/kg). A slit lamp emitting blue-greenargon laser irradiation (Haag-Streit, Köniz, Switzerland) was used totreat the right eye of the anesthetized rat with 80 to 120 applicationsdirected toward three of the four episcleral veins and toward 270° ofthe limbal plexus. The laser beam was applied with a power of 1 W for0.2 seconds, producing a spot size of 100 μm at the episcleral veins and50 μm at the limbal plexus. At a second laser session 1 week later, thesame parameters were used, except that the spot size was 100 μm in allapplications. Irradiation was directed toward all four episcleral veinsand 360° of the limbal plexus (Schori et al., 2001).

(iii) Induction of acute high intraocular pressure. An increase in IOPwas achieved in the right eyes of deeply anesthetized rats (ketaminehydrochloride 50 mg/kg, xylazine hydrochloride 0.5 mg/kg, injectedintramuscularly) by inserting a 30-gauge needle connected to apolyethylene tube and a bag of normal saline (0.9%) infusion. Theinfusion bag was placed 1 meter above the rat's head, creating a closedloop circulation. High IOP was induced for exactly 1 hour after whichthe needle was removed from the eye and the hole was self-sealed.

(iv) Measurement of intraocular pressure. Most anesthetic agents cause areduction in IOP (Jia et al., 2000b), thus precluding reliablemeasurement. To obtain accurate pressure measurements while the rat wasin a tranquil state, we injected the rat intraperitoneally (i.p.) with10 mg/mL acepromazine, a sedative drug that does not reduce IOP, andmeasured the pressure in both eyes 5 minutes later with a tonometer(Tono-Pen XL; Automated Ophthalmics, Ellicott City, Md., USA), afterapplying Localin to the cornea. Because of the reported effect ofanesthetic drugs on IOP measured by Tono-Pen (Jia et al., 2000b),pressure was always measured at the same time after injection ofacepromazine, and the average of 10 measurements taken from each eye wasrecorded. Measurements were taken on different occasions (every 2 daysfor 3 weeks), all at the same time of day. The untreated contralateraleye served as the control.

(v) Anatomical assessment of retinal damage caused by the increase inIOP. The hydrophilic neurotracer dye dextran tetramethylrhodamine(Rhodamine Dextran; (Molecular Probes, Eugene, Oreg., USA) was applieddirectly into the intra-orbital portion of the optic nerve. Only axonsthat survive high IOP and remain functional with live cell bodies cantake up the dye and demonstrate labeled RGCs. The rats were killed 24hours later, and their retinas were excised, whole mounted, andpreserved in 4% paraformaldehyde. The RGCs were counted undermagnification of ×800 in a fluorescence microscope (Carl Zeiss,Oberkochen, Germany). From each retina four fields were counted, allwith the same diameter (0.076 mm²) and at the same distance from theoptic disc (Kipnis et al., 2001; Yoles et al., 2001). Eyes fromuntreated rats were used as a control. RGCs were counted by an observerwho was blinded to the identity of the retinas.

(vi) Active immunization with Cop 1 and adjuvant. Rats with eitherlaser-induced increase in IOP or acute IOP elevation were immunized withCop 1 (100 μg) (Teva Pharmaceutical Industries Ltd., Petach Tikva,Israel) emulsified in 2.5 mg/ml of CFA in a total volume of 100 μl(Kipnis et al., 2000). Each animal was vaccinated subcutaneously at theroot of the tail.

(vii) Active immunization with Cop 1 and no adjuvant. Cop-1 in PBS wasgiven at different concentrations and at different time points after theprimary insult subcutaneously. Topical administration of Cop-1 was doneafter immersing the substance in PBS at a concentration of 20 mg/ml.Since each drop was of 50 μl, we administered 1 drop every 5 minutes fora total of 5 drops in 25 minutes.

Example 1 Cop-1 Vaccination Protects RGCs From IOP-Induced Death WhenGiven Without Vehicle

Previous studies have shown that Cop 1 emulsified in an adjuvantprotects against IOP-induced RGC death. Here we examined whether theeffect is long-lasting in a chronic model.

Animals were subjected to unilateral elevation of IOP and immunized onthe day of laser treatment (to induce IOP elevation). Rats weresubjected to chronic elevation of IOP on the day of the first laserirradiation. Animals were divided into 4 groups: two groups receivedCop-1 emulsified in CFA and two groups received PBS in CFA. From onegroup of Cop-1-treated animals retinas were excised 3 weeks later andthe second group received Cop-1 2, 6 and 9 weeks latter. From thisgroup, retinas were excised 12 weeks after the first laser irradiation.Of the two PBS-CFA-treated groups, one was analyzed for RGC survival 3weeks after the first laser irradiation, and one received additionalinjection of PBS-CFA at 2, 6 and 9 weeks after the laser, and theretinas were excised 12 weeks after the first laser. The results areshown in FIG. 1. Assessment of the number of surviving RGCs 3 and 12weeks later revealed a significant difference between the control andCop-1 immunized and the PBS immunized rats, even 12 weeks followinginitiation of IOP elevation. Beneficial effect of Cop-1 (expressed as %of RGC death) was seen at 3 weeks (12.6±5 vs. 45.7±8, n=7) and at 12weeks (33.7±1.4 vs. 57.2±6.3, n=5 and 4, respectively). Thus, 12 weeksafter the first laser irradiation there was further loss of RGCsrelative to 3 weeks (from 45.7% at 3 weeks to 57.20% by 12 weeks). Yetthe vaccination reduced the loss at 12 weeks after the first laser to33.7%. It seems that some loss was inevitable but some was amenable forprotection even as long as 12 weeks after IOP was first elevated.

Example 2 Cop-1 Immunization Without Adjuvant

Since Cop-1 is a high molecular weight compound with multiple epitopes,we considered the possibility that it might be immunogenic even withoutadjuvant.

In a first experiment, rats were subjected to IOP elevation and receivedsubcutaneous injection of different dosages of adjuvant-free Cop-1 atvarious dosages (100, 250, 500 and 1000 μg) on the first day of lasertreatment; control animals received PBS. The results are depicted inFIG. 2A and show that the optimal effect could be achieved with 500 μgof Cop-1 injected subcutaneously to the rat; higher dosage or lower wereless effective. The group treated with 500 μg showed the highest effect:26.6±10% of RGC death as compared to 44±6% of RGC death in the grouptreated with 100 μg and 50.5±8% of RGC death in the group treated with250 μg. In all groups 4-6 animals were included.

In another experiment, we also examined the timing and the frequency andfound that injection on day 7 rather than on day 0 after IOP elevationwas more effective, whereas repeated injection either at intervals ofone week or daily, were less effective Rats were subjected to IOP andreceived 500 μg of adjuvant-free Cop-1 either immediately after thefirst laser (0), or a week later (7) or on both day 0 and day 7 (0,7) ordaily for 7 consecutive days starting on day 0 (0-7). The control groupreceived PBS. Retinas were excised 3 weeks later. The results aredepicted in FIG. 2B and show that the % of RGC death was lowest in thegroup that received a single injection on day 7 and highest in the groupthat received daily injections for 7 days (19.1±4.7 vs. 41.4±6, p<0.01).

The effect of adjuvant-free Cop-1 was also tested 6 weeks after IOP wasfirst elevated. It was found that two injections—one on day 0 and thesecond one 3 weeks later—were more effective than a single injectionafter 3 weeks, suggesting that the vaccination should be synchronizedwith the kinetic of death, otherwise it is less effective. Since thedeath starts earlier than 3 weeks after IOP is first elevated, it isless effective if the first injection is given 3 weeks after IOP isfirst elevated.

Example 3 Cop-1 Effect is T-Cell Dependent

To verify that the effect of Cop-1 is indeed immune-mediated and doesnot act as a local drug, we administered Cop-1 to animals deprived of Tcells in which IOP was elevated.

In a first experiment, normal adult rats and adult rats deprived of Tcells (as a result of thymectomy at birth) were subjected to elevatedIOP. Immediately after IOP elevation, animals received Cop-1, a singleinjection, or PBS, or daily brimonidine. Three weeks later retinae wereexcised and RGCs were counted. A lower percentage of RGCs died innon-thymectomized animals than in thymectomized animals following IOPelevation (34±3 vs. 50.2±3, p<0.001, n=6 and 5, respectively). As shownin FIGS. 3A and 3B, in the thymectomized animals treated with theα2-adrenoreceptor agonist glaucoma drug brimonidine, the loss of RGCswas lower than in the non-treated thymectomized or in the Cop-1-treatedthymectomized rats [38±7 (n=6) vs. 55±5.2 (n=5); p=0.001]. The effect ofbrimonidine and Cop-1 in non-thymectomized animals with elevated IOP isshown in FIG. 3B. Treatment in non-thymectomized animals with Cop-1 wasmore effective than brimonidine (23.5±5.7 vs. 34.5±3.51, p<0.05). The %of death in the control non-treated group was 47.9±7.5. No synergybetween the two treatments was evident.

Thus, as expected, loss in T cell-deprived animals was higher than innormal animals (50% vs. 30%). Secondly, Cop-1 did not reduce IOP-inducedRGC loss in T cell-deprived animals (55% vs. 50%). In contrast, thea2-adrenoreceptor agonist used as a positive control, protected from RGCloss even in thymectomized animals (FIG. 3A). We also tested whetherthere is a synergy between the α2-adrenoreceptor agonist brimonidine andCop-1 in protecting RGCs from IOP-induced death in normal animals. FIG.3B shows that there is no additive or synergy between the two compoundsas far as the extent of RGC protection when given simultaneously. Theseresults thus show that Cop-1 is a strong immunogen and can protect RGCsfrom IOP-induced death following a single injection 7 days after IOP iselevated.

Example 4 Cop-1 Eye Drops Protect RGCs in a Model of Chronic IOP

Since Cop-1 therapy is T cell-mediated and we have shown above that asingle injection without adjuvant is sufficient to display protection,we explored now the possibility of using Cop-1 as eye drops withoutadjuvant. Assuming that approximately 10% of the eye drops get into theblood, we applied drops equivalent to 5 mg Cop-1 (ten-fold the optimal500 μg found to be active when given subcutaneously). The Cop-1 eyedrops were given either immediately (FIG. 4A) or 7 days (FIG. 4B) afterIOP elevation in a chronic model. The Cop-1 protection with the eyedrops was as effective as when given subcutaneously, when assessed 3weeks after pressure elevation (p<10⁻⁵; 103 RGCs vs. 150 RGCs).

Example 5 Cop-1 Eye Drops Protect RGCs from IOP-Induced Death in a Modelof Acute IOP

The results of the chronic IOP encouraged us to examine whether Cop-1can protect from IOP-induced loss of RGCs in an acute model of glaucoma.

We have previously established a well-calibrated model of a transientelevation of IOP (1 hour for 40 mm/Hg) which resulted 2 weeks later inan approximately 50% loss of RGCs. Application of Cop-1 without adjuvantin this acute model of IOP elevation resulted 2 weeks later in loss ofonly 20%.

Using the acute IOP elevation model in Lewis rats with an average IOP of54.75±11 mmHg, we vaccinated subcutaneously (FIG. 5A) or topically (eyedrops; FIG. 5B) with either Cop-1 or vehicle (PBS). A total of 5 mg ofCop-1 was given to each animal during a course of 25 minutes immediatelyafter an hour of elevated IOP. Average death rate two weeks after IOPelevation was 58.58±7.42 (n=4) in the control group and 31.12±3.22 inthe Cop-1 vaccinated group (n=4) p<0.001).

It appeared that the two routes of administration were similarlyeffective in protecting RGCs, as assessed two weeks after the IOP wasraised. To prove that the eye drops provide a route of immunization andnot a way of local drug application, we elevated the pressure in one eyeand applied Cop-1 in eye drops to the contralateral side. The sameeffect as when given ipsilaterally was obtained.

REFERENCES

Aharoni R., Teitelbaum D., Arnon R., Sela M. Copolymer 1 inhibitsmanifestation of graft rejection. Transplantation 27, 598 (2001)

Bakalash, S., Kipnis, J., Yoles, E. & Schwartz, M. Resistance of retinalganglion cells to an increase in intraocular pressure isimmune-dependent. Invest. Ophthalmol. Vis. Sci. 43, 2648-2653 (2002)

Fridkis-Hareli M, Neveu J M, Robinson R A, Lane W S, Gauthier L,Wucherpfennig K W, Sela M, Strominger J L. Binding motifs of copolymer 1to multiple sclerosis- and rheumatoid arthritis-associated HLA-DRmolecules. J Immunol. 162(8):4697-4704 (1999)

Fridkis-Hareli M, Santambrogio L, Stern J N, Fugger L, Brosnan C,Strominger J L. Novel synthetic amino acid copolymers that inhibitautoantigen-specific T cell responses and suppress experimentalautoimmune encephalomyelitis. J Clin Invest 109(12):1635-1643 (2002)

Hauben, E., Nevo, U., Yoles, E., Moalem, G., Agranov, E., Mor, F.,Akselrod, S., Neeman, M., Cohen, I. R., and Schwartz, M. Autoimmune Tcells as potential neuroprotective therapy for spinal cord injury.Lancet 355:286-287 (2000)

Jia, L., Cepurna, W. O., Johnson, E. C. & Morrison, J. C. Patterns ofintraocular pressure elevation after aqueous humor outflow obstructionin rats. Invest. Ophthalmol. Vis. Sci. 41, 1380-1385 (2000a)

Jia, L., Cepurna, W. O., Johnson, E. C. & Morrison, J. C. Effect ofgeneral anesthetics on IOP in rats with experimental aqueous outflowobstruction. Invest. Ophthalmol. Vis. Sci. 41, 3415-3419. (2000b)

Kipnis, J. & Schwartz, M. Dual Action of Glatiramer Acetate (Cop-1) as aTreatment for Autoimmune Diseases and a Vaccine for ProtectiveAutoimmunity after CNS Injury. Trends Mol. Med 8,319-323 (2002)

Kipnis J, Yoles E, Porat Z, Cohen A, Mor F, Sela M, Cohen I R, SchwartzM. T cell immunity to copolymer 1 confers neuroprotection on the damagedoptic nerve: possible therapy for optic neuropathies. Proc. Natl. Acad.Sci. USA 97, 7446-7451. (2000)

Kipnis J, Yoles E, Schori H, Hauben E, Shaked I, Schwartz M. Neuronalsurvival after CNS insult is determined by a genetically encodedautoimmune response. J Neurosci. 21, 4564-4571. (2001)

Levkovitch-Verbin H, Quigley H A, Martin K R, Valenta D, Baumrind L A,Pease M E. Translimbal laser photocoagulation to the trabecular meshworkas a model of glaucoma in rats. Invest. Ophthalmol. Vis. Sci. 43,402-410. (2002)

Mizrahi, T., Hauben, E. & Schwartz, M. The tissue-specific self-pathogenis the protective self-antigen: The case of uveitis. J Immunol. 169,5971-5977 (2002)

Moalem, G., Leibowitz-Amit R, Yoles E, Mor F, Cohen I R, Schwartz M.Autoimmune T cells protect neurons from secondary degeneration aftercentral nervous system axotomy. Nat. Med. 5, 49-55 (1999)

Mukibi-Muka G, Jones R C, Kibenge F S. Serological response and virusshedding of chickens inoculated with reovirus via different routes. ResVet Sci 37(2):227-9 (1984)

Russell P H, Mackie A. Eye-drop DNA can induce IgA in the tears and bileof chickens. Vet Immunol Immunopathol 80(3-4):327-32 (2001)

Schlegel P G, Aharoni R, et al. A synthetic random copolymer withpromiscuous binding to class II MHC molecules inhibits T-cellproliferative response to major and minor histocompatibility antigens invitro and confers the capacity to prevent murine graft-versus-hostdisease in vivo. Proc. Natl. Acad. Sci. USA. 93, 5061 (1996)

Schori, H., J. Kipnis, E. Yoles, E. WoldeMussie, G. Ruiz, L. A. Wheeler,and M. Schwartz. Vaccination for protection of retinal ganglion cellsagainst death from glutamate cytotoxicity and ocular hypertension:implications for glaucoma. Proc. Natl. Acad. Sci. USA 98, 3398-3403.(2001)

Schori, H., Lantner, F., Shachar, I. & Schwartz, M. Severeimmunodeficiency has opposite effects on neuronal survival inglutamate-susceptible and -resistant mice: Adverse effect of B cells. JImmunol. 169, 2861-2865(2002)

Schwartz, M., Belkin, M., Yoles, E. & Solomon, A. Potential treatmentmodalities for glaucomatous neuropathy: neuroprotection andneuroregeneration. J Glaucoma 5, 427-432 (1996)

Schwartz, M. & Kipnis, J. Multiple sclerosis as a by-product of thefailure to sustain protective autoimmunity: A paradigm shift. TheNeuroscientist 8, 405-413 (2002)

Schwartz, M. & Yoles, E. Neuroprotection: a new treatment modality forglaucoma? Curr. Opin. Ophthalmol. 11, 107-111 (2000a)

Schwartz, M. & Yoles, E. Self-destructive and self-protective processesin the damaged optic nerve: implications for glaucoma. Invest.Ophthalmol. Vis. Sci. 41, 349-351 (2000b)

Sharma J M. Introduction to poultry vaccines and immunity. Adv. Vet.Med. 41, 481-94 (1999)

Yoles E, Hauben E, Palgi O, Agranov E, Gothilf A, Cohen A, Kuchroo V,Cohen I R, Weiner H, Schwartz M. Protective autoimmunity is aphysiological response to CNS trauma. J Neurosci. 21, 3740-3748. (2001)

1. An eye-drop vaccine for therapeutic immunization of a mammalcomprising an active agent selected from the group consisting ofCopolymer 1, a Copolymer 1-related peptide, and a Copolymer 1-relatedpolypeptide.
 2. An eye-drop vaccine according to claim 1 for treatingneuronal degeneration caused by an injury, disease, disorder orcondition in the central nervous system (CNS) or peripheral nervoussystem (PNS), for preventing or inhibiting neuronal secondarydegeneration which may otherwise follow a primary injury in the CNS, forpromoting nerve regeneration in the CNS or in the PNS after injury ordisease, disorder or condition or for protecting CNS and PNS cells fromglutamate toxicity.
 3. An eye-drop vaccine according to claim 2, whereinsaid injury is spinal cord injury, blunt trauma, penetrating trauma,brain coup or contrecoup, hemorrhagic stroke, or ischemic stroke.
 4. Aneye-drop vaccine according to claim 2, wherein said disease, disorder orcondition is a senile dementia including Alzheimer's disease, aParkinsonian syndrome including Parkinson's disease, facial nerve(Bell's) palsy, Huntington's chorea, a motor neuron disease includingamyotrophic lateral sclerosis, a prion disease includingCreutzfeldt-Jakob disease, Alper's disease, Batten disease, Cockaynesyndrome, Lewy body disease, status epilepticus, carpal tunnel syndrome,intervertebral disc herniation, vitamin deficiency such as vitamin Bdeficiency, seizure disorders such as epilepsy, psychotic disorders suchas schizophrenia and anxiety, amnesia, hyperalgesia, oxidative stress,opiate tolerance and dependence, an autoimmune disease, a peripheralneuropathy associated with a disease such as amyloid polyneuropathy,diabetic neuropathy, uremic neuropathy, porphyric polyneuropathy,hypoglycemia, Sjogren-Larsson syndrome, acute sensory neuropathy,chronic ataxic neuropathy, biliary cirrhosis, primary amyloidosis,obstructive lung diseases, acromegaly, malabsorption syndromes,polycythemia vera, IgA and IgG gammapathies, complications of variousdrugs such as nitrofurantoin, metronidazole, isoniazid and toxins suchas alcohol or organophosphates, Charcot-Marie-Tooth disease, ataxiatelangiectasia, Friedreich's ataxia, adrenomyeloneuropathy, giant axonalneuropathy, Refsum's disease, Fabry's disease, lipoproteinemia,non-arteritic optic neuropathy, age-related macular degeneration, aretinal disorder such as retinal degeneration, or a disease associatedwith abnormally elevated intraocular pressure such as glaucoma.
 5. Aneye-drop vaccine according to claim 4, wherein said autoimmune diseaseis multiple sclerosis.
 6. An eye-drop vaccine according to claim 1,wherein said vaccine comprises the active agent without an adjuvant. 7.An eye-drop vaccine according to claim 1, wherein said vaccine comprisesthe active agent with a soluble adjuvant.
 8. An eye-drop vaccineaccording to claim 7, wherein said soluble adjuvant is a cytokine.
 9. Aneye-drop vaccine according to claim 8, wherein said cytokine is IL-2,IL-12, IFN-γ or GM-CSF.
 10. An eye-drop vaccine according to claim 1,wherein said active agent is Copolymer
 1. 11. An eye-drop vaccineaccording to claim 1, wherein said active agent is a Copolymer 1-relatedpeptide or a Copolymer 1-related polypeptide.
 12. An eye-drop vaccineaccording to claim 1, for administration at a frequency of at least onceevery day or every alternate day to a multiple sclerosis patient.
 13. Aneye-drop vaccine according to claim 1, for administration periodicallyat a frequency of at least once every seven days, to at least once everymonth to at least once every 2-3 months, to a non-multiple sclerosispatient.
 14. An eye-drop vaccine according to claim 13, foradministration to a glaucoma patient. 15-29. (canceled)
 30. A method oftherapeutic immunization for treating neuronal degeneration caused by aninjury, disease, disorder or condition in the central nervous system(CNS) or peripheral nervous system (PNS), for preventing or inhibitingneuronal secondary degeneration which may otherwise follow a primaryinjury in the CNS, for promoting nerve regeneration in the CNS or in thePNS after an injury, disease, disorder or condition or for protectingCNS and PNS cells from glutamate toxicity, which comprises immunizing anindividual in need with an eye-drop vaccine comprising an active agentselected from the group consisting of Copolymer 1, a Copolymer 1-relatedpeptide, and a Copolymer 1-related polypeptide, in an amount effectiveto treat, prevent or inhibit said neuronal degeneration caused by saidinjury, disease, disorder or condition in the individual.
 31. A methodaccording to claim 30, wherein said injury is spinal cord injury, blunttrauma, penetrating trauma, brain coup or contrecoup, hemorrhagicstroke, or ischemic stroke.
 32. A method according to claim 30, whereinsaid disease is a senile dementia including Alzheimer's disease, aParkinsonian syndrome including Parkinson's disease, facial nerve(Bell's) palsy, Huntington's chorea, a motor neuron disease includingamyotrophic lateral sclerosis, a prion disease includingCreutzfeldt-Jakob disease, Alper's disease, Batten disease, Cockaynesyndrome, Lewy body disease, status epilepticus, carpal tunnel syndrome,intervertebral disc herniation, vitamin deficiency such as vitamin Bdeficiency, epilepsy, amnesia, anxiety, hyperalgesia, psychosis,seizures, oxidative stress, opiate tolerance and dependence, anautoimmune disease, or a peripheral neuropathy associated with a diseasesuch as amyloid polyneuropathy, diabetic neuropathy, uremic neuropathy,porphyric polyneuropathy, hypoglycemia, Sjogren-Larsson syndrome, acutesensory neuropathy, chronic ataxic neuropathy, biliary cirrhosis,primary amyloidosis, obstructive lung diseases, acromegaly,malabsorption syndromes, polycythemia vera, IgA and IgG gammapathies,complications of various drugs such as nitrofurantoin, metronidazole,isoniazid and toxins such as alcohol or organophosphates,Charcot-Marie-Tooth disease, ataxia telangiectasia, Friedreich's ataxia,adrenomyeloneuropathy, giant axonal neuropathy, Refsum's disease,Fabry's disease, lipoproteinemia, non-arteritic optic neuropathy,age-related macular degeneration, a retinal disorder such as retinaldegeneration, or a disease associated with abnormally elevatedintraocular pressure such as glaucoma.
 33. A method according to claim32, wherein said autoimmune disease is multiple sclerosis.
 34. A methodaccording to claim 30, which comprises immunization with the activeagent without an adjuvant.
 35. A method according to claim 30, whichcomprises immunization with the active agent with a soluble adjuvant.36. A method according to claim 35, wherein said soluble adjuvant is acytokine.
 37. A method according to claim 36, wherein said cytokine isIL-2, IL-12, IFN-γ or GM-CSF.
 38. A method according to claim 30,wherein said active agent is Copolymer
 1. 39. A method according toclaim 30, wherein said active agent is a Copolymer 1-related peptide ora Copolymer 1-related polypeptide.
 40. A method according to claim 30,wherein said vaccine is administered at a frequency of at least onceevery day or every alternate day to a multiple sclerosis patient.
 41. Amethod according to claim 30, wherein said vaccine is administeredperiodically at a frequency of at least once every seven days, to atleast once every month to at least once every 2-3 months, to anon-multiple sclerosis patient.
 42. A method of therapeutic immunizationof a glaucoma patient which comprises administering to the patient aneye-drop vaccine comprising Copolymer 1 in an amount effective to treatglaucoma in said patient.
 43. A method for reducing neuronaldegeneration caused by the neurodegenerative effects of a disease, orfor reducing secondary neuronal degeneration that follows the primaryneuronal degeneration of an injury, in the central nervous system (CNS)or peripheral nervous system (PNS) of an individual in need thereof,which comprises immunizing the individual in need with an eye-dropvaccine comprising an active agent selected from the group consisting ofCopolymer 1, a Copolymer 1-related peptide, and a Copolymer 1-relatedpolypeptide, in an amount effective to reduce said neuronal degenerationcaused by injury or disease in said individual.